DNA Isolation and Restriction Enzyme Analysis Quiz

Study Notes

Proteinase K aids in digesting proteins that may be bound to DNA, facilitating its release during extraction.
Salt stabilizes DNA by neutralizing the negative charges on the phosphate backbone, aiding in its precipitation.
DNA separation from other molecules occurs through cell lysis, followed by the selective precipitation of DNA using alcohol.
Sodium dodecyl sulfate (SDS) is a surfactant that disrupts cell membranes, lysing cells and releasing their contents, including DNA.

After forming a white precipitate, the DNA is typically washed, dried, and then resuspended in a suitable buffer for further analysis.

Purpose of agarose gel electrophoresis is to separate DNA fragments based on size for analysis, visualization, or further manipulation.
Ethidium bromide intercalates with DNA, allowing visualization of the DNA bands under UV light, indicating the presence and size of DNA.
The loading buffer contains tracking dyes, often bromophenol blue or xylene cyanol, which help monitor the migration of DNA during electrophoresis.

The size of DNA molecules significantly affects their movement through agarose gel; smaller fragments migrate faster than larger ones.
A comb is inserted into the gel cast to create wells, which hold the DNA samples during electrophoresis and prevent them from diffusing.

Agarose concentration determines the gel's porosity; lower concentrations allow for larger DNA fragments to pass, while higher concentrations impede their migration.
Keeping restriction enzymes on ice or in thermoresistant containers prevents premature activity and ensures specificity in the digestion process.

Typically, two microfuge tubes are used: one for the DNA and the other for the restriction enzyme and buffer mix.
Incubating the reaction mixture at 65°C activates some enzymes and enhances efficiency, promoting the digestion process.

'Star activity' refers to the non-specific activity of restriction enzymes under suboptimal conditions, potentially leading to unwanted cuts in DNA.
Running DNA in an agarose gel after a restriction digest is critical for assessing digestion efficiency, fragment lengths, and validating the results.

Multiple restriction enzymes may be used in a digest scenario to target different recognition sites or to create specific fragment combinations essential for cloning or analysis.